Hepatitis c dsrna effector molecules, expression constructs, compositions, and methods of use

ABSTRACT

The present invention provides agents, compositions, constructs and methods for silencing HCV polynucleotides, as well as methods and compositions for treating or preventing HCV infection in a mammalian cell. In one aspect, the present invention provides an agent or composition comprising at least one double-stranded RNA effector molecule or complex. The double-stranded RNA effector molecule or complex comprises: (1) a sequence of at least 19 nucleotides having at least 90% identity with a nucleotide sequence within HCV Conserved Region 1 (SEQ ID NO: 2), HCV Conserved Region 2 (SEQ ID NO: 3), HCV Conserved Region 5 (SEQ ID NO: 4), (ATR)-1 (SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQ ID NO: 88), ATR-4 (SEQ ID NO: 89); and (2) its complementary sequence. In another aspect, the present invention provides a construct suitable for replication in a host cell, and/or suitable for expression of an RNA molecule or complex of the invention in vitro or in vivo. In a third aspect, the present invention provides a method for silencing HCV RNA in a mammalian cell, which comprises administering to the mammalian cell an agent, composition, or construct of the invention in a manner and amount effective to silence HCV RNA in the cell. In a related aspect, the invention provides a method for treating or preventing HCV infection in a patient, comprising administering to the patient an effective amount of an agent, composition, or construct of the invention as described herein.

PRIORITY

This application claims priority to U.S. Provisional Application No.60/929,335, filed Jun. 22, 2007, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to nucleic acid-based therapeutics fortreating or preventing Hepatitis C Virus (HCV) infection, andparticularly RNAi-based therapeutics.

BACKGROUND OF THE INVENTION

Hepatitis C is an RNA virus containing a single-stranded positive-senseRNA genome of about 9,600 nts. The genes for the viral structural andnon-structural proteins are flanked by 5′ and 3′ untranslated regions(UTRs), which are essential for genome replication. For example, the 5′UTR contains an internal ribosome entry site (IRES) which isindispensable for the initiation of HCV polyprotein translation. HCV hasbeen classified into six major genotypes, each comprising furthersubgroups, which differ in their sequence homology by more than 30%. Thedistribution of these genotypes differs geographically. For example,genotypes 1a and 1b are the most prevalent genotypes found within theU.S., while genotypes 2 and 3 are more prevalent in other countries.

Because of the sequence variability of HCV, the development of vaccinesand therapeutic drugs, including RNAi-based therapeutics, that would beactive against the majority of viruses, must take advantage of the rareconserved epitopes and sequences found among the viral genotypes andquasispecies. In fact, the mutability of HCV is such that even within aninfected individual, the HCV virus exists as a swarm of variants or“quasispecies” of a predominant type rather than as a single entity.

Thus, to apply a gene-silencing-based strategy to the treatment orprevention of HCV infection, it is necessary to identify sufficientlyconserved stretches of nucleotide sequence in this highly mutable virus.That is, since RNA interference is a sequence-specific effect,therapeutic or prophylactic RNAi molecules must be specific for HCVtarget sequences, despite the fact that hepatitis C viral genomes arehighly variable. While HCV target sequence conservation is an importantconsideration in the design of sequence-specific anti-HCV prophylacticor therapeutic modalities such as RNAi or antisense, e.g., some of thehighly conserved regions of the HCV genome such as the 5′ UTR are knownto be highly structured, while some regions of the viral genome arepresent in the infected cell in association with proteins which makethem largely inaccessible to antisense or RNAi. The lack of a readilyavailable HCV animal model and problems with various HCV cell culturemodels, e.g., the absence or deficiencies in viral infection orreplication models, have hindered the development of anti-HCVpharmaceuticals of all types.

Despite well over a decade of research efforts, there are no vaccinesavailable for HCV. As a consequence, the rate of new HCV infectionsaround the world is extremely high. The WHO estimates that globally 170million individuals carry chronic HCV infections and that new infectionsare established at a rate of 3 to 4 million annually.

Chronic HCV infection induces liver inflammation, causing progressiveliver disease that can lead to cirrhosis and hepatocellular carcinoma(liver cancer). Chronic HCV infection becomes established in 75%-85% ofindividuals experiencing an initial infection, and HCV-related liverfailure is the most common indication cited for liver transplantation inthe U.S. Chronic HCV infection in its early stages may cause only mildnon-specific symptoms, such as fatigue, or be completely asymptomatic,leaving many infected individuals unaware that they carry a dangerouschronic infection.

Current therapies for HCV infection, which may include a 6 to 12 monthregimen of pegylated interferon and ribavirin, can lead to a cure in aminority of patients. Response rates vary by HCV genotype, with genotype2 and 3 patients exhibiting a 76% response rate to the current standardtherapy while patients infected with genotype 1a and 1b having only a46% response rate. Unfortunately, genotype 1 accounts for 60% of globalinfections and is the dominant strain in the U.S., Japan, and WesternEurope. Complicating genotype 1 resistance to ribavarin and interferonis the fact that both drugs have side effect profiles that can requiredose reduction or discontinuance of therapy when patients experienceside effects. Further complicating patient outcomes is the fact thatpatients who fail an initial treatment regimen rarely respond favorablyto a subsequent round of treatment with interferon and ribavarin.

Clinicians who treat HCV patients are hopeful that current and futureresearch programs will yield options that improve the response rate forgenotype 1 patients, which is currently less than 50% using ribavarinand interferon. New treatment options that have a more tolerable sideeffect profile would improve patient compliance and enable more patientsto complete a full course of therapeutic intervention.

There remains a need for treatment options for HCV-exposed or infectedpatients, including for highly conserved nucleic acid-based molecules,including double-stranded RNAs and constructs encoding dsRNAs, capableof inhibiting the replication of HCV in mammalian cells. Such nucleicacid based anti-HCV therapeutic agents have the potential to improvepatient response rates to therapy, improve adverse event profile, andeliminate or significantly delay the development of drug resistantescape mutant virus.

SUMMARY OF THE INVENTION

The present invention provides agents, compositions, constructs, andmethods for silencing HCV polynucleotides, as well as methods andcompositions capable of inhibiting HCV replication and for treating orpreventing HCV infection in a mammalian cell.

In one aspect, the present invention provides an agent or compositionfor silencing HCV RNA in a cell. In one aspect, the agent or compositioninhibits HCV viral replication and antigen expression in a mammaliancell, preferably a human cell. In this aspect, the agent or compositioncomprises at least one double-stranded RNA effector molecule or complex.The double-stranded RNA effector molecule or complex comprises: (1) asequence of at least 19, 20, or 21 consecutive nucleotides having atleast 90%, 95%, or 100% identity with a nucleotide sequence within HCVConserved Region 1 (SEQ ID NO: 2), HCV Conserved Region 2 (SEQ ID NO:3), HCV Conserved Region 5 (SEQ ID NO: 4), active target region (ATR)-1(SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQ ID NO: 88), or ATR-4(SEQ ID NO: 89); and (2) its complementary sequence. Preferably thedsRNA effector molecule will include (1) a sequence of 19 to 29, 19 to25, 20 to 25, 21 to 25, or 21 to 23 consecutive nucleotides within HCVConserved Region 1 (SEQ ID NO: 2), HCV Conserved Region 2 (SEQ ID NO:3), HCV Conserved Region 5 (SEQ ID NO: 4), active target region (ATR)-1(SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQ ID NO: 88), or ATR-4(SEQ ID NO: 89); and (2) its complementary sequence. The effectormolecule may optionally form a stem-loop structure, with the sequence ofat least 19 nucleotides and its complementary sequence being connectedvia a loop sequence, thereby providing, for example, short-hairpin RNAs(shRNAs) suitable for RNAi-based HCV therapeutics. In one aspect,multiple different of such dsRNA effector molecules of the invention,e.g., two, three, four, five or more, are administered to or expressedconcomitantly in a mammalian cell, to eliminate or substantially delaythe emergence of drug resistant viral escape mutants.

In another aspect, the present invention provides a construct suitablefor replication in a host cell, and/or suitable for expression of an RNAmolecule of the invention in vitro or in vivo. The construct of theinvention encodes at least one RNA effector molecule of the invention,which may be operably linked to a promoter sequence, such as an RNAPolymerase I, RNA polymerase II, or RNA polymerase III promoter sequenceas described herein. In one aspect, multiple such dsRNA effectormolecules of the invention, e.g., hairpin dsRNA molecules, are encodedby a single expression construct under the control of one or more ofsuch promoters.

In a third aspect, the present invention provides a method for silencingor inhibiting the replication of HCV, including inhibition of HCV RNAand/or HCV antigen expression, in a mammalian cell. In this aspect, themethod of the invention comprises administering to the mammalian cell anagent, composition, or construct of the invention in a manner and amounteffective to inhibit HCV replication and/or HCV RNA or antigenexpression in the cell. In a related aspect, the present inventionprovides a method for treating or preventing HCV infection in a patient,comprising administering to the patient an effective amount and regimenof an agent, composition, or construct of the invention as describedherein.

The present invention further provides methods for preparing the dsRNAeffector molecules, compositions, and constructs of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a number of highly active shRNAs which map to four activetarget regions (ATR-1, ATR-2, ATR-3, and ATR-4) within the HCV 5′ UTR.

FIG. 2 is a plasmid diagram of the multi-cistronic plasmid QJ, whichexpresses four different active shRNAs, HCV 5′-21-61, HCV 5′-21-94, HCV5′-21-124, and HCV 5′-21-135.

FIG. 3 is a plasmid diagram of a mono-cistronic plasmid HCV 5′ 21-61,which expresses the shRNA HCV 5′ 21-61 from the 7SK 4A promoter.

DETAILED DESCRIPTION OF THE INVENTION

To identify sequences that are most conserved among HCV genomesworldwide, a bioinformatic analysis was conducted. There are 93 completegenomes published in GenBank version 134.0 and these were compared forthe identification of sequences of 19 nts or greater that are >95%conserved, and which could potentially serve as target sites for smallinhibitory RNAs (siRNAs) and short-hairpin RNAs (shRNAs). The followingsequences were identified within the HCV 5′UTR, and are shown withrespect to GenBank Accession ID AB047639 (SEQ ID NO: 1).

HCV Conserved region 1: nts 35-102 of AB047639. (SEQ ID NO: 2)5′-atcactcccctgtgaggaactactgtcttcacgcagaaagcgcctagccatggcgttagtatgagtgt-3′HCV Conserved region 2: nts 119-176 of AB047639. (SEQ ID NO: 3)5′-ccccccctcccgggagagccatagtggtctgcggaaccggtgagtac accggaattgc-3′HCV Conserved region 5: nts 270-338 of AB047639. (SEQ ID NO: 4)5′-gcgaaaggccttgtggtactgcctgatagggcgcttgcgagtgccccgggaggtctcgtagaccgtgca-3′

Four highly conserved and highly active target regions (ATR), preferredfor some applications, were identified:

ATR-1: 5′-CCCTGTGAGGAACTACTGTCTTCACGCAGAA-3′ (SEQ ID NO: 86), mapping tonucleotide coordinates 42 to 76 of GenBank Accession No. AB047639, foundwithin Conserved Region 1 (SEQ ID NO: 2).ATR-2: 5′-TCCCGGGAGAGCCATAGTGGTCTGCGGAA-3′ (SEQ ID NO: 87), mapping tonucleotide coordinates 126 to 154 of GenBank Accession No. AB047639,found within Conserved Region 2 (SEQ ID NO: 3).ATR-3: 5′-CGAAAGGCCTTGTGGTACTGC-3′, (SEQ ID NO: 88) mapping tonucleotide coordinates 271 to 297 of GenBank Accession No. AB047639,found within Conserved Region 5 (SEQ ID NO: 4).ATR-4: 5′-TGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCA-3′, (SEQ ID NO: 89) mappingto nucleotide coordinates 305 to 338 of GenBank Accession No. AB047639,found within Conserved Region 5 (SEQ ID NO: 4).

In this context, the present invention provides agents, compositions,constructs and methods for silencing HCV polynucleotides, as well as fortreating or preventing HCV infection in a mammalian cell.

Effector RNA Molecules and Complexes

In one aspect, the present invention provides an agent for silencing HCVRNA in a cell. The agent comprises at least one double-stranded RNAeffector molecule or complex, which comprises: (1) a sequence of atleast 19, 20, or 21 consecutive nucleotides having at least about 90%identity with a nucleotide sequence within HCV Conserved Region 1 (SEQID NO: 2), HCV Conserved Region 2 (SEQ ID NO: 3), HCV Conserved Region 5(SEQ ID NO: 4), ATR-1 (SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQID NO: 88), or ATR-4 (SEQ ID NO: 89) and (2) its complementary sequence.

In this context, the nucleotide “t” in SEQ ID NOS: 1, 2, 3, 4, etc. isconsidered to be identical with “u,” which would take the place of “t”in the corresponding RNA sequence. Thus, throughout this application itwill be understood that where RNA sequences are described forconvenience with respect to encoding or corresponding DNA sequences, “t”will be replaced by “u” in the RNA sequence.

In certain embodiments, the at least 19 nucleotides of the dsRNAeffector molecule or complex has at least about 95% identity with anucleotide sequence within HCV Conserved Region 1 (SEQ ID NO: 2), HCVConserved Region 2 (SEQ ID NO: 3), HCV Conserved Region 5 (SEQ ID NO:4), ATR-1 (SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQ ID NO: 88),or ATR-4 (SEQ ID NO: 89), such as at least about 96%, 97%, 98%, 99%, orabout 100% identity. Identity between two nucleotide sequences may bedetermined using any suitable algorithm known in the art, such asTatusova et al., Blast 2 sequences—a new tool for comparing protein andnucleotide sequences, FEMS Microbiol Lett. 174:247-250 (1999).

In some embodiments, the at least 19 nucleotides of the dsRNA effectormolecule or complex has a sequence selected from within nucleotides42-76 (ATR-1), 126-154 (ATR-2), 271 to 297 (ATR-3), 305 to 338 (ATR-4)or 271-338 of SEQ ID NO: 1 (GenBank Accession ID AB047639). For example,the dsRNA effector molecule or complex may comprise at least 19nucleotides selected from within nucleotides 305-338 of SEQ ID NO: 1.

The dsRNA effector molecules and complexes may have double-strandedregions that vary somewhat in length, so long as the effector moleculeor complex is effective for silencing the target polynucleotide, thatis, the length of the double-stranded region is generally sufficient totrigger RNAi-mediated degradation of the target sequence. For example,the agent or composition of the invention may comprise at least onedouble-stranded RNA effector molecule or complex containing adouble-stranded region of from 19 or 21 base pairs to about 30 or 40base pairs, or from 21 base pairs to about 27 base pairs. In certainembodiments, the double-stranded RNA effector molecule or complexcontains a double-stranded region of about 21 or about 27 base pairs.

In accordance with the invention, the double-stranded RNA molecules andcomplexes of the invention may exist in a denatured or substantiallydenatured form. Alternatively, the double-stranded RNA molecules andcomplexes may exist in a double-stranded conformation, or asubstantially double-stranded conformation, or a partiallydouble-stranded conformation, at least with respect to the regions ofcomplementarity. Generally, the RNA molecules and complexes of theinvention are capable of forming double-stranded structures underphysiological conditions (e.g., intracellular conditions), where thesestructures are sufficient to trigger RNAi-mediated gene silencing.

In some embodiments, the double-stranded RNA effector molecule orcomplex has a double-stranded region that comprises, or consistsessentially of (or consists of) in one strand, a sequence selected fromSEQ ID NOS: 5-42 as disclosed herein. Such sequences, which correspondto HCV Conserved Regions 1, 2, and 5, are listed in Table 3.

The double-stranded RNA effector molecules of the invention comprise aregion of self-complementarity such that nucleotides in one segment ofthe molecule base pair with nucleotides in another segment of themolecule (e.g., stem-loop or hairpin structure as described herein). Incontrast, the double-stranded RNA effector duplexes or complexes of theinvention include at least two separate polynucleotide strands that havea region of complementarity to each other. The double-stranded RNAcomplexes (i.e., duplexes) may be fully complementary, that is, maycontain no single stranded regions, such as single stranded ends. Inother embodiments, the double-stranded RNA complex contains shortsingle-stranded ends, such as single-stranded 5′ or 3′ ends of fromabout 1 to about 5 nucleotides (e.g., 1, 2, or 3 nucleotides).

In certain embodiments, the effector molecule is a short hairpin dsRNA(shRNA) or a microRNA. A “shRNA” (short-hairpin RNA) is an RNA moleculeof less than approximately 200 or 100 nucleotides, such as about 70nucleotides or less, in which at least one stretch of nucleotides (e.g.,at least about 19 nucleotides) is base paired with a complementarysequence located on the same RNA molecule and separated from thecomplementary sequence by an unpaired region. These single-strandedhairpin regions form a single-stranded loop between the stem structurecreated by the two regions of base complementarity. The length andnucleotide sequence of the loop sequence is not narrowly critical, andmay range, for example, from about 4 to 5 to about 20 nucleotides inlength, or from about 7 to about 10 nucleotides in length. For example,the loop sequence may be about 9 nucleotides in length. An exemplaryloop sequence is 5′-agagaactt-3′ (SEQ ID NO: 43). The loop may varyconsiderably in length and sequence, although loop sequences whichassume significant secondary structure are to be avoided, as are poly T(e.g., T₄ to T₅ or more) sequences, which might trigger prematuretermination of transcription for effector molecules expressed bypolymerase III promoters. In addition to a “stem” region which comprisesthe identified homologous and complementary HCV sequences, in certainembodiments the hairpin molecule and/or the expression vector encodingthe hairpin RNA will include additional 5′ and/or 3′ sequences,including in some embodiments 5′ and/or 3′ flanking sequences as well asloop sequences derived from miRNAs. See e.g., US 2004/0053411, theteaching of which is hereby incorporated by reference.

Exemplary shRNAs in accordance with the invention have a sequenceselected from SEQ ID NOS: 44-81. The complementary strand may also,optionally, have from one to five uracil nucleotides at its 3′-end,which may correspond to transcribed transcription termination sequences.

In addition to single shRNAs, the invention includes dual or bi-fingerand multi-finger hairpin dsRNAs, in which the RNA molecule comprises twoor more of such stem-loop structures, each separated by asingle-stranded spacer region. In some embodiments such two or morestem-loop structures may be encoded by an expression construct andoperably linked to a single promoter. Thus, the hairpin dsRNA may be asingle hairpin dsRNA or a bi-fingered, or multi-fingered dsRNA hairpinas described in PCT/US03/033466 or WO 04/035766, or a partial or forcedhairpin structure as described in WO 2004/011624, the disclosures ofwhich are hereby incorporated by reference. In these embodiments, themulti-finger hairpin RNA contains two, three, or four double strandedregions, each corresponding to an HCV target sequence independentlyselected from SEQ ID NOS: 5-42, such as SEQ ID NOS: 11, 19, 22, and 33.In these multi-finger hairpin RNAs the stem loop sequences may beindependently selected from SEQ ID NOS: 44-81.

HCV target sequences (as described above) may also be combined intoother RNA structures suitable for RNAi-based therapeutics, including asingle stem loop structure with multiple double-stranded regionsseparated by mismatch region(s). Such RNA structures are disclosed inU.S. application Ser. No. 10/531,349, which was published as US2006/0035344 on Feb. 16, 2006, which is hereby incorporated byreference. In these embodiments, the multi-target effector molecule maycomprise two, three, or four double-stranded regions each correspondingto an HCV target sequence (as described herein) independently selectedfrom SEQ ID NOS: 5-42, such as SEQ ID NO: 11, 19, 22, and 33.

The RNA molecules and complexes of the invention may be composed purelyor predominately of ribonucleotides found naturally in RNA (A, U, C, G),or may contain chemically modified nucleotides. Exemplary chemicallymodified nucleotides include phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal base” nucleotides, “acyclic” nucleotides,5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxyabasic residue incorporation. These, as well as other chemicalmodifications, support RNAi-mediated gene silencing while havingsuperior serum stability.

RNA Compositions

The invention further provides compositions containing from two to fivedouble-stranded RNA effector molecules or complexes, such as from two tofive double-stranded RNA effector molecules or complexes as describedabove. For example, the two to five double-stranded RNA effectormolecules (e.g., shRNAs) or complexes may each comprise: (1) a sequenceof at least 19 nucleotides having at least 90% identity with anucleotide sequence within Conserved Region 1 (SEQ ID NO: 2), ConservedRegion 2 (SEQ ID NO: 3), Conserved Region 5 (SEQ ID NO: 4); ATR-1 (SEQID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQ ID NO: 88), or ATR-4 (SEQID NO: 89), and (2) its complementary sequence. Where the two to fivedouble-stranded RNAs are each shRNAs, the shRNAs each further comprise aloop sequence as described above.

Thus, the composition of the invention may contain a plurality ofdouble-stranded RNA effector molecules, each having a double-strandedregion that comprises (or consists essentially of) a sequenceindependently selected from SEQ ID NOS: 5-42, and its complementarysequence. An exemplary composition may have: a first dsRNA effectormolecule comprising SEQ ID NO: 11 and its complementary sequence; asecond dsRNA effector molecule comprising SEQ ID NO: 19 and itscomplementary sequence; a third dsRNA effector molecule comprising SEQID NO: 22 and its complementary sequence; and a fourth dsRNA effectormolecule comprising SEQ ID NO: 33 and its complementary sequence. One,two, three, or four of these double stranded RNA effector molecules maybe in the form of short-hairpin RNAs, that is, the sense and anti-sensestrands may be connected by a short loop sequence as described herein.Thus, in these embodiments, the composition of the invention maycomprise from two to five (e.g., four) double-stranded RNA effectormolecules independently selected from SEQ ID NOS: 44-81. For example,the composition may comprise the four shRNAs represented by thesequences: SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 61, and SEQ ID NO:72. In some embodiments the plurality of anti-HCV RNA effector moleculesare expressed from a plasmid or viral vector within a human cell.

Alternatively still, the composition of the invention may contain aplurality of double-stranded RNA effector duplexes or complexes, eachhaving a double-stranded region that comprises (or consists essentiallyof) a sequence independently selected from SEQ ID NOS: 5-42, and itscomplementary sequence. For example: a first RNA effector complex maycomprise, in one RNA strand, the nucleotide sequence of SEQ ID NO: 11; asecond RNA effector complex may comprise, in one strand, the nucleotidesequence of SEQ ID NO: 19; a third RNA effector complex may comprise, inone strand, the nucleotide sequence of SEQ ID NO: 22; and a fourth RNAeffector complex may comprise, in one strand, the nucleotide sequence ofSEQ ID NO: 33. Such RNA molecules are base-paired in the complex with(or are capable of base-pairing with) a complementary or partiallycomplementary RNA molecule in the complex, that is an RNA moleculehaving a sequence complementary to SEQ ID NOS: 11, 19, 22, and 33,respectively.

Constructs

In another aspect, the present invention provides a construct encodingat least one RNA molecule or complex of the invention. The construct maybe, for example, a plasmid or viral vector. Such constructs may beexpression constructs suitable for expression of the encoded RNA invitro or in vivo, or may otherwise be suitable for replication of theconstruct in a host cell, such as a prokaryotic or eukaryotic host cell,including bacteria, yeast, and mammalian including human host cells. Theconstruct may include an origin of replication, mechanism for selection(e.g., antibiotic resistance gene) as well as elements to facilitateremoval of the RNA-encoding sequence for sub-cloning into additionalconstructs or vectors as may be desired, such as conveniently placedrestriction endonuclease cleavage sites (e.g., flanking the RNA-encodingsequence(s)).

In some embodiments, the construct is an expression construct containinga DNA segment that encodes an RNA molecule of the invention, with theDNA segment being operably linked to a promoter to drive expression ofthe RNA molecule. An “expression construct” is any double-stranded DNAor double-stranded RNA designed to produce an RNA of interest. Theinvention includes expression constructs in which one or more of thepromoters is not in fact operably, linked to a polynucleotide sequenceto be transcribed, but instead is designed for efficient insertion of anoperably-linked polynucleotide sequence to be transcribed by thepromoter, for instance by way of one or more restriction cloning sitesin operative association with the one or more promoters.

Transfection or transformation of the expression construct into arecipient cell allows the cell to express an RNA effector moleculeencoded by the expression construct. An expression construct may be agenetically engineered plasmid, virus, recombinant virus, or anartificial chromosome derived from, for example, a bacteriophage,adenovirus, adeno-associated virus, retrovirus, lentivirus, poxvirus, orherpesvirus. Expression vectors for use with the invention containsequences from bacteria, viruses or phages. Such vectors includechromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, bacteriophages, yeast episomes, yeastchromosomal elements, and viruses; as well as vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, cosmids and phagemids. Exemplary vectorsare double-stranded DNA phage vectors and double-stranded DNA viralvectors.

In certain embodiments, the expression construct of the invention is aplasmid, such as a plasmid suitable for delivery to and/or for RNAexpression in a mammalian cell.

The construct of the invention encodes at least one double-stranded RNAeffector molecule, which comprises: (1) a sequence of at least 19nucleotides having at least about 90% identity with a nucleotidesequence within HCV Conserved Region 1 (SEQ ID NO: 2), HCV ConservedRegion 2 (SEQ ID NO: 3), HCV Conserved Region 5 (SEQ ID NO: 4); ATR-1(SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQ ID NO: 88), or ATR-4(SEQ ID NO: 89), and (2) its complementary sequence.

In certain embodiments, the at least 19 nucleotides have at least about95% identity with a nucleotide sequence within HCV Conserved Region 1(SEQ ID NO: 2), HCV Conserved Region 2 (SEQ ID NO: 3), HCV ConservedRegion 5 (SEQ ID NO: 4), ATR-1 (SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87),ATR-3 (SEQ ID NO: 88), or ATR-4 (SEQ ID NO: 89), such as at least about96%, 97%, 98%, 99%, or about 100% identity. Identity between twonucleotide sequences may be determined using any suitable algorithmknown in the art, such as Tatusova et al., Blast 2 sequences—a new toolfor comparing protein and nucleotide sequences, FEMS Microbiol Left.174:247-250 (1999).

In some embodiments, the at least one RNA effector molecule encoded bythe construct or vector has a sequence selected from within nucleotides42-76, 126-154, or 271-338 of SEQ ID NO: 1 (GenBank Accession IDAB047639). For example, the construct may encode an RNA effectormolecule comprising at least 19 nucleotides selected from withinnucleotides 305-338 of SEQ ID NO: 1.

The expression construct of the invention may encode RNA effectormolecules having double-stranded regions that vary somewhat in length,so long as the effector molecule is effective for silencing the targetpolynucleotide. For example, the expression construct may encode atleast one double-stranded RNA effector molecule containing adouble-stranded region of from 19 or 21 base pairs to about 30 or 40base pairs, or from 21 to about 27 base pairs. In certain embodiments,the double-stranded RNA effector molecule contains a double-strandedregion of about 21 or about 27 base pairs.

In some embodiments, the construct (e.g., expression vector) encodesoneor more double-stranded RNA effector molecules, each having adouble-stranded region that comprises, or consists essentially of (orconsists of) in one strand, a sequence selected from SEQ ID NOS: 5-42.Such sequences are listed in Table 3. In some embodiments, theexpression vector encodes at least one double stranded RNA effectormolecule that comprises: 1) a sequence selected from SEQ ID NO: 9, 11,19, 22, 26, 31, 32, and 33; 2) the complement of said sequence, andoptionally. 3) a sequence linking 1) and 2). In some embodiments, theexpression vector will encode at least two, three, four, or more of suchdsRNA effector molecules. In some embodiments the expression vector willencode at least four different dsRNA effector molecules comprising, indouble stranded conformation, SEQ ID NOS: 11, 19, 22, and 33; SEQ IDNOS: 9, 11, 31, and 33; SEQ ID NOS: 11, 19, 31, and 33; SEQ ID NOS: 19,22, 32, and 33; SEQ ID NOS: 11, 19, 22, and 33; or SEQ ID NOS: 26, 31,32, and 33.

The encoded double stranded RNA effector molecule(s) may be composed oftwo separate complementary, or partially complementary RNA molecules(e.g., expressed from separate expression cassettes on the same ordifferent vectors), or may alternatively be in the form of a single RNAmolecule, such as a short-hairpin RNA. In the latter embodiment, thesense and anti-sense polynucleotides are connected via a loop sequence,which is generally single-stranded. The length of the loop sequence isnot narrowly critical, and may range, for example, from 4 to 20nucleotides in length, or from 7 to 10 nucleotides in length, or longerif desired, e.g., 30 nt, 40 nt, etc. For example, the loop sequence maybe about 9 nucleotides in length. An exemplary loop sequence is5′-agagaactt-3′ (SEQ ID NO: 43). The loop sequence may also varyconsiderably, so long as structure-forming complementarity is avoided,and, in the case of RNA polymerase III promoter transcribed sequences,poly T termination sequences are avoided.

In accordance with this aspect of the invention, exemplary expressionconstructs encode double-stranded RNA effector molecule(s) having asequence selected from SEQ ID NOS: 44-81. The complementary strand mayalso, optionally have from 1 to 5 uracil nucleotides at its 3′-end,representing transcribed RNA pol III termination signal nucleotides. Theexpressed dsRNA effector molecule may also optionally include one tofive or more additional 5′ nucleotides, which may vary depending on thetranscription start site.

The expression construct may be engineered to encode multiple, e.g.,three, four, five or more RNA molecules, such as the RNA moleculesdescribed herein, including short hairpin dsRNAs. The expressionconstruct may include a plurality of promoters, e.g., RNA polymerase I,II or III promoters, mitochondrial promoters, etc., operable in the hostmammalian cell. Each promoter may be operably linked to a sequenceencoding one or more RNA molecules of the invention, followed by anappropriate termination sequence. In addition to targeting highlyconserved HCV sequences, the ability to co-deliver two, three, four,five or more different RNA effector molecules radically reduces theability of the virus to develop escape mutants. While “cocktail”pharmaceutical preparations including multiple active components can beformulated, dsRNA expression constructs provide an attractive deliveryvehicle for accomplishing such co-delivery of a plurality of differentantiviral effector molecules by a single pharmaceutical entity. Themanufacturing and regulatory advantages of such an approach are readilyapparent.

In certain embodiments, the expression construct encodes two or moreRNAs of the invention, such as 2, 3, 4, 5, or more double stranded RNAmolecules, such as shRNAs. Thus, the expression construct may encodedouble stranded RNAs, such as shRNA hairpins, specific for one or moreof HCV Conserved Regions 1, 2 or 5, or ATR-1, ATR-2, ATR-3, or ATR-4.

In these embodiments, the invention provides one or more expressionconstructs, which collectively encode from two to five or moredouble-stranded RNA effector molecules, such as from two to five or moredouble-stranded RNA effector molecules as described herein. In certainembodiments, each expression construct encodes two, three, or fourdouble-stranded RNA effector molecules, and preferably shRNAs. Theencoding sequences, which may each be operably linked to a promoter, mayeach encode: (1) a sequence of at least 19 nucleotides having at least90% identity with a nucleotide sequence within HCV Conserved Region 1(SEQ ID NO: 2), HCV Conserved Region 2 (SEQ ID NO: 3), or HCV ConservedRegion 5 (SEQ ID NO: 4), ATR-1 (SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87),ATR-3 (SEQ ID NO: 88), or ATR-4 (SEQ ID NO: 89); (2) its complementarysequence; and optionally (3) a loop sequence.

For example, the expression construct of the invention may encode aplurality of double-stranded RNA effector molecules havingdouble-stranded regions comprising (or consisting essentially of) asequence independently selected from SEQ ID NOS: 5-42 and itscomplementary sequence. In one embodiment, the construct encodes: afirst effector molecule comprising or consisting essentially of (orconsisting of) the nucleotide sequence of SEQ ID NO: 11, a secondeffector molecule comprising or consisting essentially of (or consistingof) the nucleotide sequence of SEQ ID NO: 19, a third effector moleculecomprising or consisting essentially of (or consisting of) thenucleotide sequence of SEQ ID NO: 22, and a fourth effector moleculecomprising or consisting essentially of (or consisting of) thenucleotide sequence of SEQ ID NO: 33. One, two, three, or four of thesedouble stranded RNA effector molecules may be in the form ofshort-hairpin RNAs, that is, having sense and anti-sense strandsconnected by a short loop sequence as described herein. Thus, in theseembodiments, the expression construct of the invention may encode aplurality of dsRNA effector molecules independently selected from SEQ IDNOS: 44-81. The construct may encode two, three, or four double-strandedRNA effector molecules represented by the sequences: SEQ ID NO: 50, SEQID NO: 58, SEQ ID NO: 61, and SEQ ID NO: 72.

The expression construct may encode an RNA molecule of the invention andits complementary stand separately, that is from separate promoters(e.g., separate expression cassettes). In this embodiment, thedouble-stranded molecule is produced intracellularly upon expression ofthe RNAs and subsequent hybridization. Typically, with expressedinterfering RNA (eiRNA), the dsRNA is expressed in the first transfectedcell from an expression vector. In such a vector, the sense strand andthe antisense strand of the dsRNA may be transcribed from the samenucleic acid sequence using e.g., two convergent promoters at either endof the nucleic acid sequence or separate promoters transcribing either asense or antisense sequence. Alternatively, two plasmids can becotransfected, with one of the plasmids designed to transcribe onestrand of the dsRNA while the other is designed to transcribe the otherstrand.

In certain embodiments, the construct is an expression constructsuitable for expression (e.g., transcription) of the encoded RNA invitro or in vivo. In these embodiments, the construct encodes an RNAmolecule of the invention operably linked to a promoter sequence.Suitable promoter sequences suitable for run-off transcription in vitroare known, and include T3 and T7 promoters. In other embodiments, theexpression construct encodes an RNA molecule of the invention operablylinked to a promoter suitable for expression (e.g., transcription) ofthe RNA in a mammalian cell. The mammalian cell may be in culture, ormay be a patient's cell, e.g., a patient afflicted with or at risk ofacquiring HCV. Promoters suitable for RNA expression in a mammalian cellare known, and include those described in WO 2006/033756, which ishereby incorporated by reference. For example, the construct of theinvention may contain a DNA sequence encoding an RNA molecule of theinvention operably linked to an RNA polymerase III promoter, a 7SKpromoter, an H1 promoter, or a U6 promoter. Further, where the constructencodes a plurality of RNA molecules, each independently controlled byits own promoter, the promoters may be the same or different. In oneaspect, an expression construct may comprise two, three, four or more7SK 4A promoters as described in WO 2006/033756. Additional promotersmay be selected from an RNA polymerase I promoter, an RNA polymerase IIpromoter, a T7 polymerase promoter, an SP6 polymerase promoter, a tRNApromoter, and a mitochondrial promoter.

Generally, vector-directed expression of short RNA effector moleculesincluding short hairpin dsRNAs is most efficient when under the controlof a mammalian promoter that the host cell naturally employs forexpression of small RNA molecules. These promoters comprise the familyof RNA Polymerase III promoters, including Type 1, Type 2, and Type 3RNA Polymerase III promoters. Prototypical examples of promoters in eachclass are found in genes encoding 5s RNA (Type 1), various transfer RNAs(Type 2) and U6 small nuclear RNA (Type 3). Another promoter family(transcribed by RNA Polymerase I) is also dedicated in the cell totranscription of small structural RNAs; however, this family may be lessdiverse in sequence than the RNA Polymerase III promoters. Finally, RNAPolymerase II promoters are used in the transcription of theprotein-coding messenger RNA molecules, as distinguished from the smallstructural and regulatory RNA mentioned above. The majority of promotersystems known in the art utilize RNA Polymerase II promoters, which maynot be preferred for production of small RNAs. An exception may beshRNAs expressed by RNA polymerase II or III promoters in a miRNAcontext as taught in e.g. U.S. Ser. No. 10/429,249 and PCT/US2007/81103,which is hereby incorporated by reference. RNA polymerase IIIpromoter-based vectors containing one promoter have been described inthe art (see, e.g., U.S. Pat. No. 5,624,803, Noonberg et al., “In vivooligonucleotide generator, and methods of testing the binding affinityof triplex forming oligonucleotides derived therefrom”), and adescription of U6-based vector systems can be found in Lee et al., Nat.Biotechnol. 20:500-05 (2002). Yu et al., Proc. Natl. Acad. Sci. USA99:6047-52 (2002), describe an expression system for short duplex siRNAscomprising a T7 and U6 promoter. Miyagishi and Taira, Nat. Biotechnol.20:497-500 (2002), describe expression plasmids for short duplex siRNAscomprising expression cassettes containing tandem U6 promoters, eachtranscribing either the sense or the antisense strand of an siRNA, whichare then annealed to form duplex siRNAs.

Where it is desired to deliver short dsRNAs, multiple RNA polymerase IIIpromoter expression constructs (as taught in WO 06/033756, which ishereby incorporated by reference), may be used in accordance with theinvention. The multiple RNA polymerase III promoters may be utilized inconjunction with promoters of other classes, including RNA polymerase Ipromoters, RNA polymerase II promoters, etc. Preferred in someapplications are the Type III RNA pol III promoters including U6, H1,and 7SK, which exist in the 5′ flanking region, include TATA boxes, andlack internal promoter sequences. A preferred 7SK promoter is the 7SK 4Apromoter variant taught in WO 06/033756, the nucleotide sequence ofwhich is hereby incorporated by reference. In such expression constructseach promoter may be designed to control expression of an independentRNA expression cassette, e.g., a shRNA expression cassette. Suchmultiple RNA polymerase III promoter expression constructs are suitablefor expression of multiple, e.g., three, four, five, or more anti-HCVshRNA effector molecules of the invention. Each dsRNA effector molecule,e.g., hairpin dsRNA, may be transcribed from its own promoter or one ormore promoters may be engineered to each transcribe a single RNA strandencoding a series or “gang” of two, three or more shRNA moleculesseparated by single-stranded regions. RNA Pol III promoters may beespecially beneficial for expression of small engineered RNAtranscripts, because RNA Pol III termination occurs efficiently andprecisely at a short run of thymine residues in the DNA coding strand,without other protein factors. T₄ and T₅ are the shortest Pol IIItermination signals in yeast and mammals, with oligo (dT) terminatorslonger than T₅ being rare in mammals. Accordingly, the multiplepolymerase III promoter expression constructs of the invention may alsoinclude an appropriate oligo (dT) termination signal, i.e., a sequenceof 4, 5, 6 or more Ts, operably linked 3′ to each RNA Pol III promotercassette in the DNA coding strand. That is, a DNA sequence encoding oneor more RNA effector molecules, e.g., a dsRNA hairpin or RNA stem-loopstructure to be transcribed, is inserted between the Pol III promoterand the termination signal.

The invention provides means for delivering to a host cell sustainedamounts of 2, 3, 4, 5, or more different antiviral dsRNA hairpinmolecules (e.g., specific for 2, 3, 4, 5, or more different viralsequences or elements), in a genetically stable mode, so as to inhibitviral replication while preventing, decreasing, or delaying generationof viral escape mutants, and without evoking a dsRNA stress response. Inaccordance with this aspect, each dsRNA hairpin may be expressed from anexpression construct, and controlled by e.g. an RNA polymerase IIIpromoter. In some such embodiments a single RNA polymerase promoter,e.g. a pol II or pol III promoter, may express a plurality of dsRNAhairpins. In some such embodiments the dsRNA hairpins will be presentwithin 5′ and 3′ flanking miRNA sequences.

Thus, the expression constructs of the invention provide a convenientmeans for delivering a multi-drug regimen comprising several differentRNAs of the invention to a cell or tissue of a host vertebrate organism,thereby potentiating the anti-viral activity, and reducing thelikelihood that multiple independent mutational events will produceresistant virus. This provides an important advantage in counteringviral variation both within human and animal host populations andtemporally within a host due to mutation events.

In certain embodiments, the expression construct contains at least twoexpression cassettes, each expression cassette directing the expressionof a shRNA independently selected from SEQ ID NOS: 44-81, such as SEQ IDNOS: 50, 58, 61, and 72. Each expression cassette may independentlycomprise at least one promoter, e.g., an RNA polymerase III promoterselected from a U6 promoter, a 7SK promoter, an H1 promoter, and an MRPpromoter. For example, each expression cassette may comprise a 7SKpromoter driving the expression of at least one double-stranded RNAeffector molecule, and an RNA pol III termination signal.

Pharmaceutical Compositions

The RNAs and constructs of the invention may be formulated aspharmaceutical compositions, comprising, in addition to effectiveamounts of the RNA(s) or construct(s) necessary to produce the desiredbiological effect, pharmaceutically acceptable carriers. Such carriersmay comprise, for example, agents for facilitating the transfection ofmammalian cells, which are well known. Exemplary transfection agents andcompositions, which may be used in accordance with the presentinvention, are Lipofectamine2000™ (Invitrogen, Carlsbad, Calif.), aswell as those reviewed and described in US 2006/0084617 published Apr.20, 2006, which is hereby incorporated by reference in its entirety. Seealso, the methods and compositions for delivery of nucleic acids astaught in in WO 2006/033756, the teaching of which is herebyincorporated herein in its entirety. Nucleic acids such as shRNAs mayalso be delivered to distal organs such as the liver by transfectingskeletal muscle cells (e.g., injection, injection/electroporation, orhydrodynamic vessel delivery) with an expression vector as taught inU.S. Ser. No. 11/935,925 (PCT/US2007/83805), the teaching of which ishereby incorporated by reference in its entirely.

In various embodiments, the pharmaceutical composition includes about 1ng to about 20 mg of nucleic acid, e.g., RNA, DNA, plasmids, viralvectors, recombinant viruses, or mixtures thereof (as described above),which provide the desired amounts of the nucleic acid molecules. In someembodiments, the composition contains about 10 ng to about 10 mg ofnucleic acid, about 0.1 mg to about 500 mg, about 1 mg to about 350 mg,about 25 mg to about 250 mg, or about 100 mg of nucleic acid. Those ofskill in the art of clinical pharmacology can readily arrive atappropriate dosing schedules with routine experimentation.

Other suitable carriers include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The pharmaceutical composition generally contains one or morepharmaceutically acceptable additives suitable for the selected routeand mode of administration. These compositions may be administered by,without limitation, any parenteral route including intravenous (IV) orintra-arterial (IA) (including hydrodynamic delivery methods in whichincreasing intravessel pressure increases transfection of thesurrounding cells), intramuscular (IM), intramuscular/electroporation,subcutaneous (SC), intradermal, intraperitoneal, intrathecal, as well astopically, orally, and by mucosal routes of delivery such as intranasal,inhalation, rectal, vaginal, buccal, and sublingual. Generally, thepharmaceutical compositions of the invention are prepared foradministration to mammalian subjects including primates and humans, andare in the form of liquids, including sterile, non-pyrogenic liquids forinjection, emulsions, powders, aerosols, tablets, capsules, entericcoated tablets, or suppositories.

Methods for Inhibiting Expression of Target Polynucleotides

The invention further provides a method for silencing an HCV RNA in amammalian cell. As used herein, the term silencing of an HCV RNA refersto reducing the abundance of the target polynucleotide in the cell, orin a patient treated with a sequence-specific anti-HCV agent of theinvention, whether via RNA stability and/or via replication rate of theHCV polynucleotide, including inhibitory effects on production, levelsor persistence of positive and/or negative strand HCV RNA, includingvarious qualitative and quantitative measures of HCV viral load, e.g.,quantitative PCR, transcription-mediated amplification (TMA), andbranched DNA assay (bDNA). HCV RNA silencing also includes inhibitoryeffects on viral protein expression. The target polynucleotide (e.g.,HCV RNA) may be reduced by about ½, ⅕, 1/10, 1/100 as compared to apositive control cell (e.g., a cell infected with HCV). In someembodiments, the target polynucleotide is undetectable in cellstransfected or patients treated with an agent, composition (includingpharmaceutical composition), or construct of the invention).

In this aspect, the invention comprises administering to a mammaliancell, such as a primate or human cell infected with HCV, at least oneagent, composition, or construct of the invention, as describedpreviously herein, including small-hairpin RNAs and including theencoding expression constructs. The cell in such embodiments may be acell culture, or may be an animal model or patient. In theseembodiments, the invention provides a method of reducing levels of HCVRNA in a cell either in vitro or in vivo, as well as methods forreducing an HCV titer in vitro or in vivo. In a preferred aspect, theagent is capable of reducing HCV replication in a human hepatocyte in anin vitro HCV infection/replication model such as the recently developedinfectious cell culture systems, which more closely reflect actual humanHCV infection. The JFH-1 HCV clone, utilized in the Examples below,which is able to replicate efficiently in Huh7 cells and can secreteinfectious viral particles, represents one such improved model. Seee.g., Zhong et al., Robust hepatitis C virus infection in vitro, PNAS,102, No. 26, pp 9294-9299, (2005). See also Yi M, Villanueva R A, ThomasD L, Wakita T, Lemon S M. Production of infectious genotype 1a hepatitisC virus (Hutchinson strain) in cultured human hepatoma cells. Proc NatlAcad Sci USA 2006; 103(7): 2310-2315. In accordance with theseembodiments, the double-stranded RNA effector molecules may beintroduced into the cell by transforming or transfecting a cell with anexpression construct of the invention, or alternatively by directlyintroducing the double stranded RNA.

The present invention further provides a method for treating,ameliorating or preventing HCV infection in a patient (e.g., a patienthaving, or at risk of acquiring an HCV infection), comprisingadministering to said patient, including primates such as humans, aneffective amount of an agent, composition, or construct of theinvention, suitable for triggering RNAi-mediated degradation of thepatient. Thus, the method of the invention reduces viral replication ininfected cells, and in certain embodiments, eliminated the infection.The presence of HCV RNA in the blood is considered to be an indicationthat the virus is actively replicating (reproducing and infecting newcells). In some embodiments, HCV viral load is reduced (by 10%, 20%,50%, 75%, 90% or more) in an infected individual administered asequence-specific dsRNA of the invention. In some aspects, viral loadwill be reduced from high to low levels (less than 2 million copies/mL),desirably to undetectable levels. It is thought that individuals withviral load below 400,000 IU/mL respond better to therapeutic agents thatthose with higher levels of virus.

The present invention may, in certain embodiments, employ the methodsdisclosed in U.S. Ser. No. 11/935,925 (PCT/US2007/83805), “In VivoDelivery of Double Stranded RNA to a Target Cell”, which is herebyincorporated by reference in its entirety. Specifically, delivery intoskeletal muscle cells of expression constructs encoding dsRNA(s) mayresult in targeted inhibition of gene expression in other organs andtissues of the body such as liver. The intramuscular delivery may beachieved in a variety of ways, including needle or needleless IMinjection, IM injection/electroporation, and intravascular/hydrodynamicdelivery. Without being bound by any particular theory, delivery ofdsRNA to distal tissues such as liver cells, for example, may bemediated by extracellular vesicles (exovesicles) containing expresseddsRNA or injected siRNA or shRNA that bud from the surface oftransfected muscle cells.

The HCV treated by the method of the invention (e.g., targeted by theagents, compositions, and constructs described herein) may be anygenotype, subtype, or quasispecies, including genotypes 1a, 1b, 2a, 2b,3a, 4 and 5, and combinations thereof. In certain embodiments, the HCVinfection is non-responsive to interferon-based therapy, and/or othernucleoside analogs such as ribavirin, or other antivirals, making themethods of the invention particularly desirable. In other embodiments,the agents, compositions, and constructs are administered before,during, or after interferon therapy, where necessary to control oreliminate infection.

In certain embodiments, the agents, compositions, and constructs of theinvention are administered so as to inhibit viral replication withoutevoking a dsRNA stress response. Further, unlike traditional antiviralagents, the method of the present invention may reduce the likelihoodthat multiple independent mutational events will produce resistantvirus, thereby providing an important advantage in countering viralvariation both within human and animal host populations and temporallywithin a host due to mutation events.

Some dsRNA sequences, possibly in certain cell types and through certaindelivery methods, may result in an interferon response. The methods ofthe invention may be performed so as not to trigger an interferon/PKRresponse, for instance by using shorter dsRNA molecules between 20 to 25base pairs, by expressing dsRNA molecules intracellularly, or by usingother methods known in the art. See US Published Application20040152117, which is herein incorporated by reference. For instance,one of the components of an interferon response is the induction of theinterferon-induced protein kinase PKR. To prevent an interferonresponse, interferon and PKR responses may be silenced in thetransfected and target cells using a dsRNA species directed against themRNAs that encode proteins involved in the response. Alternatively,interferon response promoters are silenced using dsRNA, or theexpression of proteins or transcription factors that bind interferonresponse element (IRE) sequences is abolished using dsRNA or other knowntechniques.

By “under conditions that inhibit or prevent an interferon response or adsRNA stress response” is meant conditions that prevent or inhibit oneor more interferon responses or cellular RNA stress responses involvingcell toxicity, cell death, an anti-proliferative response, or adecreased ability of a dsRNA to carry out a PTGS event. These responsesinclude, but are not limited to, interferon induction (both Type 1 andType II), induction of one or more interferon stimulated genes, PKRactivation, 2′5′-OAS activation, and any downstream cellular and/ororganismal sequelae that result from the activation/induction of one ormore of these responses. By “organismal sequelae” is meant any effect(s)in a whole animal, organ, or more locally (e.g., at a site of injection)caused by the stress response. Exemplary manifestations include elevatedcytokine production, local inflammation, and necrosis. Desirably theconditions that inhibit these responses are such that not more than 95%,90%, 80%, 75%, 60%, 40%, or 25%, and most desirably not more than 10% ofthe cells undergo cell toxicity, cell death, or a decreased ability tocarry out a PTGS event, compared to a cell not exposed to suchinterferon response inhibiting conditions, all other conditions beingequal (e.g., same cell type, same transformation with the same dsRNA).

Apoptosis, interferon induction, 2′5′-OAS activation/induction, PKRinduction/activation, anti-proliferative responses, and cytopathiceffects are all indicators for the RNA stress response pathway.Exemplary assays that can be used to measure the induction of an RNAstress response as described herein include a TUNEL assay to detectapoptotic cells, ELISA assays to detect the induction of alpha, beta andgamma interferon, ribosomal RNA fragmentation analysis to detectactivation of 2′5′-OAS, measurement of phosphorylated eIF2a as anindicator of PKR (protein kinase RNA inducible) activation,proliferation assays to detect changes in cellular proliferation, andmicroscopic analysis of cells to identify cellular cytopathic effects.See, e.g., US Published Application 20040152117, which is hereinincorporated by reference.

Methods for Making Agents and Compositions

The dsRNA molecules and constructs of the invention may be made usingconventional molecular biology techniques, including standard genecloning and in vitro RNA synthesis protocols. Such methods are wellknown, and are described in Sambrook et al, Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press(1989); and Ausubel et al., ed. Current Protocols in Molecular Biology,John Wiley & Sons, Inc. (1987)). See also the methods taught in U.S.Pat. No. 6,143,527, “Chain reaction cloning using a bridgingoligonucleotide and DNA ligase”.

The following examples are provided to describe and illustrate thepresent invention. As such, they should not be construed to limit thescope of the invention. Those in the art will well appreciate that manyother embodiments also fall within the scope of the invention, as it isdescribed hereinabove and in the claims.

EXAMPLES

The following Examples are provided for illustration only.

All possible 21-mer and 27-mer expressed shRNAs were constructed basedon HCV Conserved Regions 1 (SEQ ID NO: 2), 2 (SEQ ID NO: 3) and 5 (SEQID NO: 4). Thus, for shRNAs targeting Conserved Region 1, twenty-six21-mers and sixteen 27-mers were constructed; for Conserved Region 2,thirty-nine 21-mers and thirty-three 27-mers were constructed; and forConserved Region 5, twenty-eight 21-mers and sixteen 27-mers wereconstructed.

Example 1 Silencing HCV Replication in a Viral Infection Cell CultureModel

A human liver-derived cell line such as the Huh7 cell line istransfected with an eiRNA plasmid expressing short-hairpin RNA (shRNA)molecules that comprise sequences homologous and complementary to theidentified conserved sequences in the HCV genome. Followinginternalization of the plasmid into hepatocytes and nuclearlocalization, transcription of the eiRNA plasmid from one or more RNApol III promoters results in production of the HCV-specific shRNAs.

The transfected cells are then infected with HCV JFH-1 virus. The JFH-1clone is able to replicate efficiently in Huh7 cells and can secreteinfectious viral particles. See e.g., Zhong et al., Robust hepatitis Cvirus infection in vitro, PNAS, 102, No. 26, pp 9294-9299, (2005).

Using this model, cells were transfected with various eiRNA constructsand then infected with HCV virus. The cells were then monitored for lossof HCV replication as described below.

The following is an example of an experiment that was performed usingeiRNA vectors encoding HCV sequences derived from GenBank accessionnumber AB047639. HCV sequences in these described eiRNA vectors werehighly conserved sequences identified as described elsewhere herein. Theparticular eiRNA backbone vector used for this experiment contained a7SK promoter to drive expression of the encoded RNAs. Each vectorencoded only one shRNA. The shRNA coding sequence was followed by an RNApol III termination sequence. Sequences of the 7SK promoter, RNA pol IIItermination signal, and encoded shRNAs are all shown at the end of theexample. Similar vectors containing U6 promoters (another RNA pol IIItype 3 promoter) and RNA pol III termination signals are commerciallyavailable such as the “siLentGene-2 Cloning Systems” vector fromPromega, Inc., Madison, Wis. One of ordinary skill in the art can alsocreate them according to the information provided herein.

Experimental Procedure: Transfection.

Huh7 cells cultured in DMEM medium were seeded into 96-well plates at adensity of 4×10³ cells/well. All transfections were performed the dayafter cell seeding using Lipofectamine2000™ (Invitrogen, Carlsbad,Calif.) according to the manufacturer's directions. In this experiment,cells were transfected with 200 ng of the eiRNA plasmids and wereinfected 12 hours later with HCV infectious viral clone JFH-1 at amultiplicity of infection (MOI) of 0.1 ffu/cell. DNA was heldconstant/transfection at 200 ng. In experiments where less than 200 ngof the eiRNA plasmid was transfected, the remainder of the DNA is madeup by including an inert plasmid DNA, pGL2-Basic (Promega, Madison Wis.)in amounts that brought the total DNA in the transfection to 200 ng.Prior to transfection, medium was removed from the cells and the cellswashed with phosphate-buffered saline (PBS), followed by addition of 100uL of DMEM containing 10% Fetal Bovine Serum (FBS). TheDNA/Lipofectamine2000 transfection mix was added to the cells and thesewere incubated at 37° C. for 12 hours. The medium containing thetransfection mix was then removed and the cells were washed once withPBS. At this point, 100 uL of DMEM containing 10% FBS was added to thecells. Infection with JFH-1 then commenced with the addition of thevirus at a MOI of 0.1 ffu/cell. All transfections were carried out intriplicate. Control groups were pGL2-basic alone (200 ng of pGL2-basic),a green fluorescent protein (GFP) plasmid instead of the eiRNA plasmid(200 ng pGFP), and a group that was untransfected but was infected withJFH-1 at the same MOI.

Monitoring Cells for Loss of HCV Replication.

At 48 hours following transfection, cells were monitored for the loss orreduction in HCV replication by measuring viral RNA by quantitativereverse-transcription PCR (qRT-PCR). The cells were lysed using TotalRNA Lysis Buffer (Applied Biosystems. Foster City, Calif.) and the RNAwas harvested using an ABI Prism 6100 Nucleic Acid Prep Station (AppliedBiosystems). HCV RNA was quantified by qRT-PCR using a SYBR-greenlabeled probe. The PCR primers used to amplify a 75 bp productcorresponding the 5′ UTR of the genotype 2a HCV genome (GenBankAB047639) were 5′-TCTGCGGAACCGGTGAGTA-3′ (sense) (SEQ ID NO: 82) and5′-TCAGGCAGTACCACAAGGC-3′ (antisense) (SEQ ID NO: 83). The PCR primersused to amplify a 225 bp product of the human GAPDH coding sequence(GenBank NM002046) were 5′-GAAGGTGAAGGTCGGAGTC-3′ (sense) (SEQ ID NO:84) and 5′-GAAGATGGTGATGGGATTTC-3′ (antisense) (SEQ ID NO: 85). HCV andGAPDH transcript levels were determined relative to a standard curvecomprised of serial dilutions of plasmid containing the HCV cDNA or thehuman GAPDH gene. HCV copies per ug of total cellular RNA werenormalized to GAPDH transcript levels using a modified comparativethreshold cycle method ddCt (2″^((ddCt))); where ddCt is the differencebetween the Average GAPDH and the GAPDH of the individual sample. Foldreduction in HCV copy numbers was calculated as: (average number of HCVcopies per ug total cellular RNA in control transfectedsamples)+(average number of HCV copies per ug total cellular RNA ineiRNA transfected samples).

Results:

A number of the shRNA plasmids transfected led to a decrease in HCVreplication as compared to negative controls. The results in Table 1 arepresented as the average of three plates and are shown as bothfold-inhibition and the equivalent percent reduction.

A total of 135 eiRNAs (plasmid expressed shRNAs) containing 21-merdouble-stranded stem sequences and 118 eiRNAs containing 27-merdouble-stranded stem sequences were tested in the assay. The eiRNAsshowing a 4-fold or greater inhibition are listed in Table 1 along witha representative selection of eiRNAs showing less than 4-foldinhibition. The eiRNA vectors encode the HCV sequences listed in Table2. The sequences of the shRNAs are shown as well as the map coordinatesof the sense sequence (found 3′ to the underlined 9 nt loop sequence)relative to the HCV JFH-1 clone, Gen Bank accession number ABO47639 (SEQID NO: 1). The sequences of the encoded shRNAs include an antisense HCVsequence followed by the loop sequence (underlined in Table 1) followedby a second HCV sequence, which is the complement to the first HCVsequence. (It will be understood that either the antisense or sensesequence may be located 5′ to the loop sequence, i.e.,5′-antisense-loop-sense-3′ or 5′-sense-loop-antisense-3′.) The loopstructure does not need to be a fixed sequence or length, and severalloop sequences were used with no significant impact on the functioningof the eiRNA construct. The second HCV sequence is followed by a seriesof T residues, e.g., 1, 2, 3, or more Ts, preferably at least 4 or 5 Ts,that function as the termination signal for RNA pol Ill. These Tnucleotides are not included in Table 2.

TABLE 1 Fold Percent eiRNA inhibition reduction HCV 5′ 21-2 1 0 HCV 5′21-16 1 0 HCV 5′ 21-50 1 0 HCV 5′-21-55 5 80 HCV 5′-21-56 5 80 HCV5′-21-57 5 80 HCV 5′-21-61 7 85.7 HCV 5′ 21-63 4 75 HCV 5′ 21-70 1 0 HCV5′ 21-73 1 0 HCV 5′ 21-88 5 80 HCV 5′ 21-89 5 80 HCV 5′ 21-90 4 75 HCV5′ 21-92 7 85.7 HCV 5′ 21-94 9 88.9 HCV 5′-21-122 4 75 HCV 5′-21-123 475 HCV 5′-21-124 7 85.7 HCV 5′-21-125 5 80 HCV 5′-21-126 8 87.5 HCV5′-21-127 7 85.7 HCV 5′-21-128 8 87.5 HCV 5′-21-129 4 75 HCV 5′-21-130 683.3 HCV 5′-21-131 3 67 HCV 5′-21-133 12 91.7 HCV 5′-21-134 9 88.9 HCV5′-21-135 10 90 HCV 5′ 27-1 1 0 HCV 5′ 27-8 4 75 HCV 5′ 27-9 4 75 HCV 5′27-12 1 0 HCV 5′-27-16 8 87.5 HCV 5′ 27-45 1 0 HCV 5′-27-53 4 75HCV5′-27-73 1 0 HCV 5′ 27-111 1 0

nucleotide coordinates sense sequence relative Sequence (5′antisense-loop-sense 3′) GenBank No. AB047639TAGTTCCTCACAGGGGAGTGAagagaacttTCACTCCCCTGTGAGGAACTA 36-56CCGGTTCCGCAGACCACTATGagagaacttCATAGTGGTCTGCGGAACCGG 50-70TATGGCTCTCCCGGGAGGGGGagagaacttCCCCCTCCCGGGAGAGCCATA 121-141ACCACTATGGCTCTCCCGGGAagagaacttTCCCGGGAGAGCCATAGTGGT 126-146GACCACTATGGCTCTCCCGGGagagaacttCCCGGGAGAGCCATAGTGGTC 127-147AGACCACTATGGCTCTCCCGGagagaacttCCGGGAGAGCCATAGTGGTCT 128-148CCGCAGACCACTATGGCTCTCagagaacttGAGAGCCATAGTGGTCTGCGG 132-152TTCCGCAGACCACTATGGCTCagagaacttGAGCCATAGTGGTCTGCGGAA 134-154TCACCGGTTCCGCAGACCACTagagaacttAGTGGTCTGCGGAACCGGTGA 141-161TACTCACCGGTTCCGCAGACCagagaacttGGTCTGCGGAACCGGTGAGTA 144-164GCAGTACCACAAGGCCTTTCGagagaacttCGAAAGGCCTTGTGGTACTGC 271-291GGCAGTACCACAAGGCCTTTCagagaacttGAAAGGCCTTGTGGTACTGCC 272-292AGGCAGTACCACAAGGCCTTTagagaacttAAAGGCCTTGTGGTACTGCCT 274-294TCAGGCAGTACCACAAGGCCTagagaacttAGGCCTTGTGGTACTGCCTGA 275-295TATCAGGCAGTACCACAAGGCagagaacttGCCTTGTGGTACTGCCTGATA 277-297AGACCTCCCGGGGCACTCGCAagagaacttTGCGAGTGCCCCGGGAGGTCT 305-325GAGACCTCCCGGGGCACTCGCagagaacttGCGAGTGCCCCGGGAGGTCTC 306-326CGAGACCTCCCGGGGCACTCGagagaacttCGAGTGCCCCGGGAGGTCTCG 307-327ACGAGACCTCCCGGGGCACTCagagaacttGAGTGCCCCGGGAGGTCTCGT 308-328TACGAGACCTCCCGGGGCACTagagaacttAGTGCCCCGGGAGGTCTCGTA 309-329CTACGAGACCTCCCGGGGCACagagaacttGTGCCCCGGGAGGTCTCGTAG 310-330TCTACGAGACCTCCCGGGGCAagagaacttTGCCCCGGGAGGTCTCGTAGA 311-331GTCTACGAGACCTCCCGGGGCagagaacttGCCCCGGGAGGTCTCGTAGAC 312-332GGTCTACGAGACCTCCCGGGGagagaacttCCCCGGGAGGTCTCGTAGACC 313-333CGGTCTACGAGACCTCCCGGGagagaacttCCCGGGAGGTCTCGTAGACCG 314-334ACGGTCTACGAGACCTCCCGGagagaacttCCGGGAGGTCTCGTAGACCGT 315-335CACGGTCTACGAGACCTCCCGagagaacttCGGGAGGTCTCGTAGACCGTG 316-336GCACGGTCTACGAGACCTCCCagagaacttGGGAGGTCTCGTAGACCGTGC 317-337TGCACGGTCTACGAGACCTCCagagaacttGGAGGTCTCGTAGACCGTGCA 318-338GACAGTAGTTCCTCACAGGGGAGTGATagagaacttATCACTCCCCTGTGAGGAACTACTGTC 35-61GCGTGAAGACAGTAGTTCCTCACAGGGagagaacttCCCTGTGAGGAACTACTGTCTTCACGC 42-68TGCGTGAAGACAGTAGTTCCTCACAGGagagaacttCCTGTGAGGAACTACTGTCTTCACGCA 43-69TTCTGCGTGAAGACAGTAGTTCCTCACagagaacttGTGAGGAACTACTGTCTTCACGCAGAA 46-72CGCTTTCTGCGTGAAGACAGTAGTTCCagagaacttGGAACTACTGTCTTCACGCAGAAAGCG 50-76GACCACTATGGCTCTCCCGGGAGGGGGagagaacttCCCCCTCCCGGGAGAGCCATAGTGGTC 122-148GTTCCGCAGACCACTATGGCTCTCCCGagagaacttCGGGAGAGCCATAGTGGTCTGCGGAAC 129-155CAATTCCGGTGTACTCACCGGTTCCGCagagaacttGCGGAACCGGTGAGTACACCGGAATTG 149-175TCTACGAGACCTCCCGGGGCACTCGCAagagaacttTGCGAGTGCCCCGGGAGGTCTGGTAGA 305-331

TABLE 3 eiRNA Sense sequence HCV5′-21-2 tcactcccctgtgaggaacta(SEQ ID NO: 5) HCV5′-21-16 ggaactactgtcttcacgcag (SEQ ID NO: 6)HCV5′-21-50 ccccctcccgggagagccata (SEQ ID NO: 7) HCV5′-21-55tcccgggagagccatagtggt (SEQ ID NO: 8) HCV5′-21-56 cccgggagagccatagtggtc(SEQ ID NO: 9) HCV5′-21-57 ccgggagagccatagtggtct (SEQ ID NO: 10)HCV5.-21-61 gagagccatagtggtctgcgg (SEQ ID NO: 11) HCV5′-21-63gagccatagtggtctgcggaa (SEQ ID NO: 12) HCV5′-21-70 agtggtctgcggaaccggtga(SEQ ID NO: 13) HCV5′-21-73 ggtctgcggaaccggtgagta (SEQ ID NO: 14)HCV5′-21-88 cgaaaggccttgtggtactgc (SEQ ID NO: 15) HCV5′-21-89gaaaggccttgtggtactgcc (SEQ ID NO: 16) HCV5′-21-90 aaaggccttgtggtactgcct(SEQ ID NO: 17) HCV5′-21-92 aggccttgtggtactgcctga (SEQ ID NO: 18)HCV5′-21-94 gccttgtggtactgcctgata (SEQ ID NO: 19) HCV5′-21-122tgcgactgccccgggaggtct (SEQ ID NO: 20) HCV5′-21-123 gcgagtgccccgggaggtctc(SEQ ID NO: 21) HCV5′-21-124 cgagtgccccgggaggtctcg (SEQ ID NO: 22)HCV5′-21-125 gagtgccccgggaggtctcgt (SEQ ID NO: 23) HCV5′-21-126agtgccccgggaggtctcgta (SEQ ID NO: 24) HCV5′-21-127 gtgccccgggaggtctcgtag(SEQ ID NO: 25) HCV5′-21-128 tgccccgggaggtctcgtaga (SEQ ID NO: 26)HCV5′-21-129 gccccgggaggtctcgtagac (SEQ ID NO: 27) HCV5′-21-130ccccgggaggtctcgtagacc (SEQ ID NO: 28) HCV5′-21-131 cccgggaggtctcgtagaccg(SEQ ID NO: 29) HCV5′-21-132 ccgggaggtctcgtagaccgt (SEQ ID NO: 30)HCV5′-21-133 cgggaggtctcgtagaccgtg (SEQ ID NO: 31) HCV5′-21-134gggaggtctcgtagaccgtgc (SEQ ID NO: 32) HCV5′-21-135 ggaggtctcgtagaccgtgca(SEQ ID NO: 33) HCV5′-27-1 atcactcccctgtgaggaactactgtc SEQ ID NO: 34)HCV5′-27-8 ccctgtgaggaactactgtcttcacgc (SEQ ID NO 35) HCV5′-27-9cctgtgaggaactactgtcttcacgca (SEQ ID NO: 36) HCV5′-27-12gtgaggaactactgtcttcacgcagaa (SEQ ID NO: 37) HCV5′-27-16ggaactactgtcttcacgcagaaagcg (SEQ ID NO: 38) HCV5′-27-45ccccctcccgggagagccatagtggtc (SEQ ID NO: 39) HCV5′-27-53cgggagagccatagtggtctgcggaac (SEQ ID NO: 40) HCV5′-27-73gcggaaccggtgagtacaccggaattg (SEQ ID NO: 41) HCV5′-27-111tgcgagtgccccgggaggtctcgtaga (SEQ ID NO: 42)

Preferred for use in anti-HCV dsRNA effector molecules of the inventionare the following conserved and actively inhibitory HCV sequences:

HCV-5-21-56, SEQ ID NO: 9, and its complement, which may be utilized asa duplex dsRNA, or as an shRNA, such as that of SEQ. ID. NO: 48;HCV-5-21-61, SEQ. ID NO: 11, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO: 50;HCV-5-21-90, SEQ. ID NO: 17, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO: 56;HCV-5-21-94, SEQ. ID NO: 19, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO: 58;HCV-5-21-124, SEQ. ID NO:22, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO: 61;HCV-5-21-128, SEQ. ID NO: 26, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO: 65;HCV-5-21-133, SEQ. ID NO:31, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO:70;HCV-5-21-134, SEQ. ID NO: 32, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO:71;HCV-5-21-135, SEQ. ID NO:33, and its complement, which may be utilizedas a duplex dsRNA, or as an shRNA, such as that of SEQ. ID NO:72;

In preferred embodiments, two, three, four, five or more of theabove-identified anti-HCV dsRNA effector molecules are administeredconcomitantly to a human cell infected with HCV. In some aspects, anexpression construct or constructs encoding such a plurality of dsRNAeffector molecules is/are administered concomitantly to a human cellinfected with HCV. In some aspects said two or more dsRNA effectormolecules will comprise in double-stranded conformation two or moresequences selected from SEQ ID NOS: 9, 11, 17, 19, 22, 26, 31, 32, and33. In some aspects said two or more dsRNA effector molecules willcomprise two or more sequences selected from SEQ ID NOS: 48, 50, 56, 58,61, 65, 70, 71, and 72.

The actively inhibitory HCV sequences map to four conserved and highlyactive target regions (ATR) (see FIG. 1):

ATR-1: 5′-CCCTGTGAGGAACTACTGTCTTCACGCAGAA-3′ (SEQ ID NO: 86), mapping tonucleotide coordinates 42 to 76 of GenBank Accession No. AB047639, foundwithin Conserved Region 1 (SEQ ID NO: 2).ATR-2: 5′-TCCCGGGAGAGCCATAGTGGTCTGCGGAA-3′ (SEQ ID NO: 87), mapping tonucleotide coordinates 126 to 154 of GenBank Accession No. AB047639,found within Conserved Region 2 (SEQ ID NO: 3).ATR-3: 5′-CGAAAGGCCTTGTGGTACTGC-3′ (SEQ ID NO: 88), mapping tonucleotide coordinates 271 to 297 of GenBank Accession No. AB047639,found within Conserved Region 5 (SEQ ID NO: 4).ATR-4: 5′-TGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCA3′ (SEQ ID NO: 89), mappingto nucleotide coordinates 305 to 338 of GenBank Accession No. AB047639,found within Conserved Region 5 (SEQ ID NO: 4).

Example 2 Silencing HCV Replication Using a Plasmid Expressing MultipleeiRNA Molecules

Plasmids expressing multiple eiRNAs (shRNAs) (known as multi-cistronicplasmids) can be used to inhibit HCV replication in a cell culture model(as described in Example 1). In this example, sequences encoding fourdifferent shRNA molecules were sequentially cloned into a single plasmidexpression vector, each operably linked to an individual RNA polymeraseIII promoter and terminator. Each shRNA contained a different sequencehomologous and complementary to the HCV genome that was previously shownto inhibit HCV replication. By expressing several different dsRNAmolecules within an HCV infected cell or a human cell capable of HCVinfection, the eiRNA expression vector represents a multi-drug regimenhaving an enhanced ability to inhibit HCV replication and to prevent thedevelopment during therapy of HCV escape mutants.

We cloned the genes for four HCV-suppressive shRNAs into a singleplasmid, each operably linked to a different RNA polymerase III promoterand terminator. The four shRNAs were 1) shRNA SEQ ID NO: 50, whichincludes HCV 5′-21-61 (SEQ ID NO: 11) and its complement; 2) shRNA SEQID NO: 58, which includes HCV-5′-21-94 (SEQ. ID NO: 19) and itscomplement; 3) shRNA SEQ. ID NO: 61, which includes HCV 5′-21-124 (SEQ.ID NO: 22) and its complement; and 4) shRNA SEQ. ID NO: 72, whichincludes HCV 5′21-135 (SEQ. ID NO: 33) and its complement. (See HCVmulti-cistronic plasmid HCV-QJ, FIG. 2), Each shRNA sequence wasexpressed from a different copy of the RNA pol III promoter 7SK 4A,although one or more other RNA pol III type 3 promoters such as 7SK, H1and/or U6 could also be used. It may be desirable in some circumstancesto select two, three, four or more different promoters to express two,three, four or more different dsRNAs of the invention. The quad-cistronplasmid was constructed essentially as described in WO 2006/0033756 andthe 7SK 4A promoter utilized is also described therein. The eiRNAs weretranscribed following intracellular uptake of the plasmid.

In the same manner as HCV-QJ (described above), other multi-cistronicplasmids (e.g., including quad-cistron plasmids HVC-QF, HCV-QG, HCV-QH,and HCV-QK), were constructed using different combinations of eiRNAs(anti-HCV shRNAs described herein) that had each been shown to besuppressive of HCV replication in their original mono-cistronic forms(see Table 4 below).

Such a mono-cistronic HCV eiRNA expression plasmid (HCV 5′ 21-61 eiRNAplasmid, which expresses shRNA SEQ ID NO: 50, which includes HCV5′-21-61 (SEQ ID NO: 11) and its complement) is illustrated in FIG. 3.

Note that any number of combinations, e.g., two, three, four, five ormore different suppressive eiRNAs, can constitute the multi-cistroniceiRNA plasmids of the invention. While a combination of two, three, fouror more of such different dsRNA effector molecules (e.g., duplex and/orhairpin dsRNAs) may be administered concomitantly to a mammalian cell asa “cocktail” of exogenously generated RNAs, it is desirable in someaspects to express them within a target mammalian cell from amulti-cistronic plasmid as described herein.

TABLE 4 Multi- cistronic plasmidshRNA sequence and corresponding HCV sequence HCV-QF HCV 5′21-56 (SED. ID. NO: 9) and its complement, expressedas shRNA SEQ ID NO: 48 HCV 5′21-135 (SEQ. ID NO: 33) and its complement,expressed as shRNA SEQ. ID NO: 72 HCV 5′21-133 (SEQ. ID NO: 31) and its complement,expressed as shRNA SEQ. ID NO: 70 HCV 5′21-61 (SEQ. ID NO: 11) and its complement, expressedas shRNA SEQ. ID NO: 50 HCV-QGHCV-5-21-94 (SEQ. ID NO: 19) and its complement, expressedas shRNA SEQ. ID NO: 58 HCV 5′21-135 (SEQ. ID NO: 33) and its complement,expressed as shRNA SEQ. ID NO: 72 HCV 5′21-133 (SEQ. ID NO: 31) and its complement,expressed as shRNA SEQ. ID NO: 70 HCV 5′21-61 (SEQ. ID NO: 11) and its complement, expressedas shRNA SEQ. ID NO: 50 HCV-QHHCV-5-21-94 (SEQ. ID NO: 19) and its complement, expressedas shRNA SEQ. ID NO: 58HCV-5-21-134 (SEQ. ID NO: 32) and its complement,expressed as shRNA SEQ. ID NO: 71HCV-5-21-124 (SEQ. ID NO: 22) and its complement, expressedas shRNA, SEQ. ID NO: 61 HCV 5′21-135 (SEQ. ID NO: 33) and its complement,expressed as shRNA SEQ. ID NO: 72 HCV-QJHCV-5-21-94 (SEQ. ID NO: 19) and its complement, expressedas shRNA SEQ. ID NO: 58 HCV 5′21-61 (SEQ. ID NO: 11) and its complement, expressedas shRNA SEQ. ID NO: 50HCV-5-21-124 (SEQ. ID NO: 22)and its complement, expressedas shRNA, SEQ. ID NO: 61 HCV 5′21-135 (SEQ. ID NO: 33) and its complement,expressed as shRNA SEQ. ID NO: 72 HCV-QKHCV-5-21-128 (SEQ. ID NO: 26) and itscomplement,expressed as shRNA SEQ. ID NO: 65HCV-5-21-134 (SEQ. ID NO: 32) and itscomplement,expressed as shRNA SEQ. ID NO: 71 HCV 5′21-133 (SEQ. ID NO: 31) and itscomplement,expressed as shRNA SEQ. ID NO: 70 HCV 5′21-135 (SEQ. ID NO: 33) and itscomplement,expressed as shRNA SEQ. ID NO: 72

The methods that were utilized for transfection of the multi-cistronicplasmids into Huh7 cells, the subsequent infection of these cells withHCV and the quantification of HCV mRNA (as a measure of the suppressionof HCV replication) were carried out as described in Example 1 above.

Results.

The suppressive activities that were observed for each of themulti-cistronic plasmids are shown in Table 5. Not only did the use ofmultiple different dsRNA effector molecules produce a very high level ofinhibition of HCV replication, we expect that use of such combinationsof different sequence-specific inhibitors will have enhanced ability toprevent the development of HCV escape mutants during therapy.

TABLE 5 Multi-cistronic Fold- Percent plasmid inhibition reductionHCV-QF 12  91.7 HCV-QG 11  90.9 HCV-QH 20  95 HCV-QJ 28  96.4 HCV-QK 25 96

All publications, patents and patent applications discussed herein areincorporated herein by reference. While in the foregoing specificationthis invention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

1. A method for silencing an hepatitis C virus (HCV) RNA in a mammaliancell comprising administering to said cell at least one double-strandedRNA effector molecule or complex, comprising: (1) a sequence of at least19 consecutive nucleotides having at least 90% identity with anucleotide sequence within Conserved Region 1 (SEQ ID NO: 2), ConservedRegion 2 (SEQ ID NO: 3), or Conserved Region 5 (SEQ ID NO: 4); activetarget region (ATR)-I (SEQ ID NO: 86), ATR-2 (SEQ ID NO: 87), ATR-3 (SEQID NO: 88), or ATR-4 (SEQ ID NO: 89); and (2) its complementarysequence.
 2. An agent for silencing hepatitis C Virus (HCV) RNA,comprising at least four double-stranded RNA effector molecules orcomplexes that comprise, or a construct that expresses: (a) (1)sequences of at least 19 nucleotides having at least 90% identity with anucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 32, SEQ ID NO: 22, andSEQ ID NO: 33; and (2) their complementary sequences; or (b) (1)sequences of at least 19 nucleotides having at least 90% identity with anucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 22, andSEQ ID NO: 33; and (2) their complementary sequences
 3. The agent ofclaim 2, wherein the double-stranded RNA effector molecules or complexescontain a double-stranded region of from 19 to 30 base pairs.
 4. Theagent of claim 3, wherein the sequence of at least 19 nucleotides andits complementary sequence are connected via a loop sequence.
 5. Theagent of claim 1, wherein at least one double-stranded RNA effectormolecule has a sequence selected from SEQ ID NOs: 50, 58, 61, 71 and 72.6. The agent of claim 5, wherein the expression construct contains atleast two expression cassettes, each expression cassette directing theexpression of at least one double-stranded RNA effector molecule.
 7. Aconstruct for silencing a hepatitis C virus (HCV) RNA, the constructencoding at least four double-stranded RNA effector molecules orcomplexes wherein the construct comprises: (a) (1) sequences of at least19 nucleotides having at least 90% identity with a nucleotide sequenceof SEQ ID NO: 19, SEQ ID NO: 32, SEQ ID NO: 22, and SEQ ID NO: 33; and(2) their complementary sequences; or (b) (1) sequences of at least 19nucleotides having at least 90% identity with a nucleotide sequence ofSEQ ID NO: 19, SEQ ID NO: 11, SEQ ID NO: 22, and SEQ ID NO: 33; and (2)their complementary sequences.
 8. The construct of claim 7, wherein thedouble-stranded RNA effector molecules contain a double-stranded regionof from 19 to 30 base pairs.
 9. The construct of claim 8, wherein thesequence of at least 19 nucleotides and its complementary sequence areconnected via a loop sequence.
 10. The construct of claim 7, wherein atleast one double-stranded RNA effector molecule has a sequence selectedfrom SEQ ID NOs: 50, 58, 61, 71 and
 72. 11. The construct of claim 7,wherein the expression construct contains at least two expressioncassettes, each expression cassette directing the expression of at leastone double-stranded RNA effector molecule.